Compositions and methods for diagnosing or treating neutrophil-mediated inflammatory disease

ABSTRACT

Disclosed are nanoparticle compositions comprising nanoparticles prepared from denatured, cross-linked albumin and a therapeutic agent for treating a neutrophil-mediated inflammation, and methods of treating neutrophil-mediated inflammation using the compositions.

CROSS REFERENCE TO RELATED PATENTS

This application is a continuation application of U.S. patentapplication Ser. No. 15/425,528 filed Feb. 6, 2017, now U.S. Pat. No.9,872,839, which is a continuation application of U.S. patentapplication Ser. No. 14/316,036 filed Jun. 26, 2014, now U.S. Pat. No.9,561,192, which claims the benefit of U.S. Provisional Application No.61/840,597 filed Jun. 28, 2013, which is incorporated by reference inits entirety.

STATEMENT REGARDING GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under P01 HL060678, R01HL109439, K25 HL111157, and R41 HL126456 awarded by National Institutesof Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods fordiagnosing or treating neutrophil-mediated inflammatory disease.

BACKGROUND OF THE INVENTION

Neutrophil adhesion to activated endothelial cells and trans-endothelialmigration of these cells are essential events of the innate immuneresponse required for kill invading pathogens and bacterial clearance.While neutrophil recruitment into site of injury is the first-line ofdefense, excessive neutrophil infiltration and activation at the vesselwall is also the primary cause of inflammation and tissue damage.Neutrophils have been implicated in numerous inflammatory diseases suchas acute lung injury, sepsis, and ischemia-reperfusion injury.Inhibition of β2 integrins using anti-β2 integrin antibodies has beenshown to block adhesion of neutrophils to endothelial cells and preventinflammation, leading to restored vascular integrity, which indicatesthat targeting neutrophils is a useful strategy in treatingneutrophil-mediated inflammatory diseases. However, antibodies have thedisadvantage of inducing generalized loss of neutrophil bactericidalfunction by impairing the ability of circulating neutrophils to adhereand migrate to the infected site.

Clearly, there is a demand for compositions and methods that permittargeting of activated neutrophils and treatment of neutrophil-mediatedinflammatory diseases. The present invention satisfies this demand.

SUMMARY OF THE INVENTION

One object of certain embodiments of the present invention is to providenanoparticle compositions and methods for detecting or treatingneutrophil-mediated diseases.

In certain embodiments, the neutrophil-mediated disease is aninflammatory disease. In certain embodiments, the disease is a vascularinflammatory disease.

In certain embodiments, the compositions include nanoparticles preparedfrom denatured, cross-linked albumin. In certain embodiments, thealbumin is denatured by desolvation. In certain embodiments, desolvationis performed using an alcohol. In certain embodiments, the alcohol isethanol.

In certain embodiments, the albumin is cross-linked usingglutaraldehyde.

In certain embodiments, the albumin nanoparticles have a mean particlediameter of about 100 nm±10 nm.

In certain embodiments, the nanoparticle composition includes apharmaceutically acceptable excipient.

In certain embodiments, a therapeutic agent or a detectable moiety isincorporated within the nanoparticles or covalently conjugated to thesurface of the nanoparticles.

In certain embodiments, the therapeutic agent is an anti-inflammationagent. In certain embodiments, the anti-inflammation agent is selectedfrom anti-inflammatory glucocorticoids, NF-kB inhibitors, p38MAP kinaseinhibitors, Syk/Zap kinase inhibitors, and siRNA oligonucleotidestargeting a molecule in activated neutrophils, or any combinationthereof.

In certain embodiments, the anti-inflammation agent is dexamethasoneand/or piceatannol.

In certain embodiments, the detectable moiety is a fluorescent moiety ora chromogenic moiety.

In certain embodiments is provided a method of treating aneutrophil-mediated disease or condition in a subject in need oftreatment by contacting activated neutrophils in the subject with ananoparticle composition that contains nanoparticles prepared fromdenatured, cross-linked albumin and a therapeutic agent incorporatedwithin or covalently attached to the nanoparticles.

In certain embodiments, the method involves treating aneutrophil-mediated inflammatory disease. In certain embodiments, themethod involves contacting activated neutrophils with a nanoparticlecomposition comprising nanoparticles containing or conjugated to ananti-inflammation agent.

In certain embodiments, the method involves treating aneutrophil-mediated inflammatory disease selected from sepsis,myocardial infarction, acute lung injury, stroke andischemia-reperfusion injury.

In certain embodiments is provided a method of detecting or monitoringneutrophil-mediated diseases comprising contacting neutrophils with ananoparticle composition that contains nanoparticles prepared fromdenatured, cross-linked albumin and a detectable moiety incorporatedwithin or covalently attached to the nanoparticles, and detecting uptakeof the detectable moiety by activated neutrophils.

The present invention and its attributes and advantages will be furtherunderstood and appreciated with reference to the detailed descriptionbelow of presently contemplated embodiments, taken in conjunction withthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The preferred embodiments of the invention will be described inconjunction with the appended drawings provided to illustrate and not tothe limit the invention, where like designations denote like elements,and in which:

FIG. 1 is a graph showing the percentage of neutrophils and monocytestreated or untreated with TNF-α that internalized albumin nanoparticles.

FIG. 2 is a graph showing the percentage of neutrophils internalizingvarious types of nanoparticles or Cy5-labeled native albumin.

FIG. 3 is a graph showing uptake of albumin nanoparticles by wild-type,FcγRIII^(−/−), Mac-1^(−/−), and LFA-1^(−/−) mice.

FIG. 4A is a graph showing adherent and rolling neutrophils/field as afunction of time in mice treated with albumin nanoparticles withpiceatannol.

FIG. 4B is a graph showing adherent and rolling neutrophils/field as afunction of time in mice treated with albumin nanoparticles withoutpiceatannol.

FIG. 5 is a graph showing round and spread adherent neutrophils treatedwith albumin nanoparticles or albumin nanoparticles incorporatingpiceatannol.

FIG. 6 is an immunoblot and graph showing phosphorylation of Syk inlysates of unstimulated neutrophils and neutrophils stimulated withTNF-α and treated with albumin nanoparticles or albumin nanoparticlesincorporating piceatannol.

FIG. 7A is a graph of lung myeloproxidase (MPO) activity in lungs ofmice with LPS-induced acute lung inflammation before and afterintravenous infusion of piceatannol-loaded albumin nanoparticles.

FIG. 7B is a graph of the number of neutrophils sequestered in lungs ofmice with LPS-induced acute lung inflammation before and afterintravenous infusion of piceatannol-loaded albumin nanoparticles; and

FIG. 7C shows the concentration of leukocytes in bronchoalveolar lavage(BAL) after intravenous infusion of albumin nanoparticles (Alb-Nano) orpiceatannol-loaded albumin nanoparticles (Pic-Alb Nano).

FIG. 8 is a graph of lung myeloproxidase (MPO) activity in lungs of micewith LPS-induced acute lung inflammation treated with piceatannol aloneor piceatannol-loaded albumin nanoparticles.

DETAILED DESCRIPTION

Disclosed herein are nanoparticle compositions suitable for treating ordetecting neutrophil-mediated diseases or conditions, includingneutrophil-mediated inflammatory diseases. Nanoparticle compositionsinclude denatured, cross-linked albumin and a therapeutic agent or adetectable moiety.

In certain embodiments, the nanoparticle compositions may include apharmaceutically acceptable excipient, vehicle, or carrier.

“Treating” or “treatment” as used herein includes inhibiting a diseaseor disorder, i.e., arresting its development, relieving a disease ordisorder, i.e., causing regression of the disorder; slowing progressionof the disorder, and/or inhibiting, relieving, or slowing progression ofone or more symptoms of the disease or disorder in a subject. Subjectstreated may include human and non-human individuals, including warmblooded animals such as mammals afflicted with, or having the potentialto be afflicted with one or more neutrophil-mediated diseases ordisorders, including neutrophil-mediated inflammatory diseases.

In certain aspects, the disclosure provides a pharmaceutical compositioncomprising the nanoparticle of the disclosure together with one or morepharmaceutically acceptable excipients, carriers, or vehicles, andoptionally other therapeutic and/or prophylactic components, asdescribed in detail in U.S. Provisional Application No. 61/840,597.

An effective amount of a nanoparticle composition is an amount effectiveto provide the desired biological result. That result can be reductionand/or alleviation of the signs, symptoms, or causes of a disease, orany other desired alteration of a biological system. An appropriate“effective” amount in any individual case can be determined by one ofordinary skill in the art using routine experimentation.

In certain embodiments, nanoparticle compositions may be prepared asdescribed below. In certain embodiments, the nanoparticles incorporatepiceatannol, a spleen tyrosine kinase (Syk) inhibitor that blocks‘outside-in’ β2 integrin signaling in leukocytes. Real-time intravitalmicroscopy of inflamed post-capillary venules of live mice, the primarysite of neutrophil adhesion and extravasation in the circulation,demonstrated that albumin nanoparticles are internalized by activatedneutrophils through endocytosis that is in part mediated by Fcγ receptorIII (FcγRIII). Mice treated with albumin nanoparticles incorporatingpiceatannol showed markedly reduced neutrophil adhesion and migrationacross the endothelium. A mouse model of endotoxin-induced acute lunginjury mediated by the infiltration of neutrophils also showed thattreatment with piceatannol-incorporated albumin nanoparticles preventslung injury.

In certain embodiments, stable albumin nanoparticles were prepared bydesolvation of albumin, for example, using ethanol, followed by albumincross-linking using a cross-linking agent such as glutaraldehyde. As oneof skill in the art will appreciate, any suitable albumin may be used inthe practice of the invention, including, but not limited to, BSA, humanserum albumin and ovalbumin. Any suitable cross-linking agent may beused to crosslink the albumin. To study internalization properties ofalbumin nanoparticles by phagocytes, fluorescent dyes were incorporatedinto nanoparticles. Transmission electron microscopy and dynamic lightscattering demonstrated that the size of albumin nanoparticles with andwithout fluorescent dyes was similar, with a mean diameter of 100±10(SD) nm. It is envisioned that albumin nanoparticle preparations havinga particle diameter in the range of 50 nm to 300 nm may also be used inthe practice of the invention.

Real-time fluorescence intravital microscopy was used to study theuptake of albumin nanoparticles by neutrophils. Vascular inflammationwas induced by intrascrotal injection of the pro-inflammatory cytokinetumour necrosis factor (TNF-α) in mice. At 3 hr post-TNF-α challenge,cremaster muscle was exposed, and neutrophils adherent to activatedvenular endothelial cells were monitored. Intravenous injection ofCy5-loaded albumin nanoparticles resulted in the nanoparticles beinglargely internalized by the leukocytes adherent to the inflamed venularendothelial cells, and to some extent by neutrophils slowly rollingalong the vessel wall. However, nanoparticles were not internalized bythe TNF-α activated endothelium itself. To confirm that thenanoparticles were primarily internalized by neutrophils, an Alexa Fluor488-labeled anti-mouse Gr-1 antibody and Cy5-loaded albuminnanoparticles were simultaneously infused. Anti-mouse Gr-1 antibody andalbumin nanoparticles showed marked co-staining. In control experiments,non-immune isotype control antibody, IgG did not show a signal. Toaddress whether albumin nanoparticles can also be internalized byunstimulated neutrophils in the circulation, Cy5-loaded albuminnanoparticles were infused intravenously. Unstimulated neutrophils didnot take up Cy5-loaded albumin nanoparticles, indicating that onlyadherent neutrophils were able to internalize the nanoparticles.Nanoparticle internalization by adherent monocytes, another phagocyticcell involved in inflammation, was evaluated. Adherent monocytes, unlikeneutrophils, did not internalize albumin nanoparticles.

To investigate determinants of nanoparticle internalization, uptake ofthree different types of nanoparticles by activated neutrophils werecompared. In the first two, BSA nanoparticles were made byethanol-induced albumin desolvation to denature albumin, followed byalbumin cross-linking to form stable particles. Albumin nanoparticleswere then either incorporated with fluorescent dye (Cy5-loaded albuminnanoparticles) or chemically conjugated to Alexa Fluor 647 bycarboxyl-amine reaction that forms covalent bonds between Alexa Fluor647 and albumin nanoparticles (Alexa Fluor 647-conjugated albuminnanoparticles). Additionally, albumin-conjugated polystyrenenanoparticles were prepared by coating yellow-green fluorescencepolystyrene nanoparticles having a diameter of 100 nm with native BSA.Uptake of these two types of albumin nanoparticles by Gr-1 positiveneutrophils following intravenous infusion at 3 hr after intrascrotalinjection of TNFα was compared. Alexa Fluor 647-conjugated albuminnanoparticles were found to be internalized by the adherent neutrophilsand showed characteristic punctual distribution in the cytosol, whereasCy5-loaded albumin nanoparticles showed diffuse fluorescence throughoutthe cell. The latter observation was attributed to the release of Cy5dye bound non-covalently to nanoparticles following nanoparticleinternalization. The punctual structures in the cytosol representedindividual or aggregated nanoparticles, presumably into lyso-endosomalcompartments. Conjugation of Cy5 to albumin nanoparticles also exhibitedthe same punctual structures in adherent neutrophils as Alexa Fluor647-conjugated albumin nanoparticles; thus, dye conjugation methodprevents dye dispersal following nanoparticle internalization. Based ona comparison of the fluorescence intensities of internalized Cy5-loadednanoparticles with Cy5-conjugated nanoparticles, uptake efficiency oftwo types of albumin nanoparticles was found to be similar. Further, thegeneral morphology of the adherent neutrophils internalizing either typeof nanoparticle was found to be the same, and cells had similar surfacearea of 54±6 (mean±SD) μm₂. Unlike nanoparticles made from denaturedalbumin, native albumin-conjugated polystyrene nanoparticles remainedbound to the surface of neutrophils without being internalized. Nativealbumin conjugated to Cy5 was not taken up by neutrophils. As quantifiedby multiple images, 95% of all adherent neutrophils similarlyinternalized either dye-loaded or dye-conjugated albumin nanoparticles.In contrast, neither albumin-conjugated polystyrene particles norCy5-conjugated albumin was internalized by the adherent neutrophils.

Because FcγRs activate endocytosis by binding IgG-opsonized particlesand denatured proteins, whether nanoparticles made of denatured albumincould be internalized through FcγR signaling was evaluated Neutrophilsobtaining from FcγRIII^(−/−) mice exhibited significantly reduced uptakeof albumin nanoparticles compared to wild-type (WT). By measuringfluorescence intensity of Cy5-loaded albumin nanoparticles perneutrophil, a distribution of nanoparticle uptake per neutrophil wasobtained. Based on this, FcγRIII was found to contribute to ˜50% oftotal uptake of albumin nanoparticles, consistent with the role of FcγRsignaling as a mechanism of immune complex internalization byneutrophils. The basis of residual uptake is unclear but may involveother Fcγ receptors. Macrophage antigen-1 (Mac-1 or αMβ2 integrin) andlymphocyte function-associated antigen-1 (LFA-1 or αLβ2 integrin)mediate neutrophil adhesion during vascular inflammation and denaturedalbumin binds to Mac-1 and may contribute to uptake of denaturedproteins; however, deletion of Mac-1 and LFA-1 had no effect on uptakeof albumin nanoparticles.

Albumin nanoparticles loaded with piceatannol, a Syk inhibitor, wereevaluated for the ability to reverse TNF-α-mediated firm adhesion ofneutrophils to venular endothelial cells, and thus mitigateinflammation. Syk signaling is crucial in the mechanism of ‘outside-in’integrin signaling that mediates β2 integrin-dependent neutrophiladhesion, spreading, and migration. Piceatannol selectively inhibits Sykactivity but because of low solubility in water, has not been effectiveas an anti-inflammatory agent. Intravenous infusion ofpiceatannol-loaded albumin nanoparticles, 1 mg/kg body weight ofpiceatannol (50 μM), significantly reduced the number of adherentneutrophil and concomitantly increased the number of rolling cells. Incontrols, albumin nanoparticles alone had no effect.

To investigate further the mechanisms of action of piceatannol-loadedalbumin nanoparticles, a flow chamber assay was used in which mouseneutrophils interacted with a monolayer of TNF-α-activated mouse lungendothelial cells under shear condition. In control experiments,Cy5-loaded albumin nanoparticles were internalized by Gr-1 positiveneutrophils as in mouse venular studies above. The nanoparticle signalwas slightly increased in neutrophils stimulated withN-formyl-methionyl-leucyl phenylalanine (fMLF). However, treatment ofneutrophils with piceatannol-loaded albumin nanoparticles, 800 μg/ml(200 μM as piceatannol) under shear conditions markedly reduced β2integrin-mediated neutrophil spreading and migration acrossTNF-α-activated endothelial cells and inhibited neutrophil adhesion.Thus, piceatannol-loaded albumin nanoparticles functioned by inhibitingβ2 integrin-mediated ‘outside-in’ signaling through Syk signaling,consistent with the known function of Syk. Syk phosphorylation infMLF-stimulated neutrophils was also abrogated in neutrophilsinternalizing piceatannol-loaded albumin nanoparticles indicating thatthe drug loaded into nanoparticles blocked Syk activity. A3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)assay was performed to eliminate any possible drug toxicity effect.Incubating neutrophils with piceatannol-loaded albumin nanoparticles,800 μg/ml (200 μM as piceatannol), did not affect cell viability. Thus,piceatannol-loaded albumin nanoparticles inhibited Syk phosphorylation,thereby impairing β2 integrin-mediated ‘outside-in’ signaling requiredfor neutrophil spreading and migration.

Excessive accumulation of neutrophils in lungs is a major factor in thepathogenesis of acute lung injury associated with sepsis. β2integrin-dependent neutrophil adhesion to lung endothelial cellscontributes to acute lung injury. Therefore, piceatannol-loaded albuminnanoparticles were evaluated for the ability to ameliorate lungneutrophil infiltration induced by intraperitoneal injection oflipopolysaccharide (LPS, 10 mg/kg body weight). Treating mice withpiceatannol-loaded albumin nanoparticles, 4.3 mg/kg body weight aspiceatannol (200 μM in blood), 2 hr after LPS challenge, markedlyreduced lung tissue myeloperoxidase (MPO) activity, an indication ofreduced neutrophil sequestration. Infiltration of both neutrophils andmonocytes determined by bronchoalveolar lavage (BAL) was also reduced bytreating mice with piceatannol-loaded albumin nanoparticles (4.3 mg/kgbody weight as piceatannol). Comparison of MPO activity in LPS-inducedlung inflammation after administration of piceatannol-loaded albuminnanoparticles and piceatannol (free drug) alone also showed thatpiceatannol-loaded nanoparticles were far more efficacious thanpiceatannol alone. Because albumin nanoparticles are preferentiallyinternalized by neutrophils in vivo, inhibition of neutrophilinfiltration in lungs by piceatannol-loaded albumin nanoparticlesappears to contribute to reduced monocyte infiltration. These resultsalso showed the utility of albumin nanoparticle loading of piceatannolfor the treatment of acute lung injury.

In summary, an albumin nanoparticle approach for the delivery of drugsinto inflammatory neutrophils adherent to endothelial cells has beendeveloped that is in part dependent on FcγRIII signaling. Adhesion ofneutrophils to activated endothelial cells was required for theinternalization of albumin nanoparticles. Because FcγRs are highlyexpressed in adherent neutrophils during inflammation and vasculardiseases, nanoparticles made of denatured albumin can be used to targetinflammatory neutrophils while sparing the essential host-defensefunction of circulating neutrophil. This strategy limits the undesirableeffects of globally blocking the essential bactericidal function ofneutrophils.

Further, the results reported herein demonstrate the feasibility ofusing albumin nanoparticles to target activated neutrophils withoutconjugating ligands or antibodies to the nanoparticle surface. Theresults hence provide the proof-of-concept of a novel nanoparticle-basedtherapeutic approach for targeting activated neutrophils to treat arange of inflammatory disorders. This represents a departure from theapproach typically taken for delivering therapeutics to desired cells,based on coating of ligands and antibodies to the nanoparticle surface.

Methods

Preparation of albumin nanoparticles loaded with fluorescent dyes orpiceatannol.

Bovine serum albumin (BSA) nanoparticles were prepared by thedesolvation technique (Weber et al., Int. J. Pharm. 196, 197-200 (2000),which is incorporated by reference herein). BSA was first dissolved at aconcentration of 20 mg/ml in deionized water. Nanoparticles were made bycontinuous addition to 1 ml of BSA (20 mg/ml) of 3.5 ml of ethanol understirring (650 rpm) for 10 min at RT. In some experiments, 1 ml BSA (20mg/ml) was mixed and incubated with 40 μl of Cy5 dye (5 mg/ml) inchloroform for 1 hr, followed by desolvation with ethanol. To makepiceatannol-loaded albumin nanoparticles, 1 ml of 20 mg/ml of BSAsolution was incubated with 1 mg of piceatannol dissolved in DMSO for 1hr. To form stable albumin nanoparticles with or without a dye orpiceatannol, BSA molecules were cross-linked by adding 20 μl of 0.2%glutaraldehyde in the suspension. The suspension was stirred overnightat RT. Nanoparticle suspension was centrifuged at 14,000 rpm for 20 minat 4° C. After drying albumin nanoparticles, 80-90% albumin was obtainedto form the nanoparticles. The nanoparticle pellet was re-suspended inwater or phosphate-buffered saline, pH 7.4 for the study.

To measure the efficiency of piceatannol incorporation in BSAnanoparticles, different concentrations of piceatannol were added in theinitial albumin solution. After centrifugation of suspension ofpiceatannol-loaded BSA nanoparticles at 14,000 rpm at 4° C. for 30 min,the supernatant (containing unbound piceatannol) was collected andcentrifuged using Microcon (10-kD cut-off) to separate unboundpiceatannol from free BSA molecules. Piceatannol molecules werequantified by measuring the absorbance at 328 nm. The loading yield ofpiceatannol in albumin nanoparticles was calculated by the followingequation: Loading yield (%) =(Drug used-unloaded drug)/Drug used. Afterdrying and weighting piceatannol-loaded albumin nanoparticles, 10-14 wt% of piceatannol was loaded in albumin nanoparticles.

To conjugate covalently Alexa Fluor-647 or Cy5 dye to albuminnanoparticles, carboxyl-amine reaction between dye and albumin was used.After albumin nanoparticles were made using the desolvation method asdescribed above, Alex Fluor 647-NHS was mixed with albuminnanoparticles, and incubated the mixture for 2 hr at RT. Albuminnanoparticles were centrifuged to remove free dye molecules to obtainAlexa Fluor 647-conjugated albumin nanoparticles.

Real-Time Fluorescence Intravital Microscopy

In vivo intravital microscopy was performed as described¹³. TNF-α (0.5μg in 250 μl saline) was intrascrotally injected into wild-type (WT) orknockout mice. At 3 hr after TNF-α injection, mice were anesthetizedwith intraperitoneal injection of the mixture of ketamine (80 mg/kg),xylazine (2 mg/kg), and acepromazine (2 mg/kg) and maintained at 37° C.on a thermo-controlled rodent blanket and a tracheal tube was inserted.After excision of the scrotum, cremaster muscles were exteriorized onthe intravital microscope table. The cremaster preparation wassuperfused with thermo-controlled (37° C.) and aerated (95% N2, 5% CO2)bicarbonate-buffered saline during the experiment. Fluorescently-labeledalbumin nanoparticles (100 μg) in 100 μl of saline were infused througha cannula placed in a jugular vein, followed by infusion of the AlexaFluor 488-labeled anti-mouse Gr-1 antibody (0.05 μg/g body weight).Rolling and adherent neutrophils were monitored in an area of 0.02 mm²in inflamed cremaster muscle venules. Images were recorded using anOlympus BX61W microscope with a 60×/1.0 NA water immersion objectivelens and a high speed camera (Hamamatsu C9300) connected to anintensifier (Video Scope International). To study the therapeuticeffects of piceatannol-loaded albumin nanoparticles, the Alexa Fluor488-labeled anti-mouse Gr-1 antibody was intravenously infused, followedby infusion of either piceatannol-loaded albumin nanoparticles orcontrol albumin nanoparticles (150 μg of albumin nanoparticlescontaining 50 μM piceatannol). At 30 or 60 minutes after infusion ofnanoparticles, rolling and adherent neutrophils were monitored. In someexperiments, an Alexa Fluor 488-labeled anti-mouse F4/80 antibody (2μg/mouse) was infused to monitor monocytes. Neutrophils that remainedstationary or did not exceed displacement of >8 μm for at least 30 secwere considered adherent. To quantify neutrophil rolling and adherence,each vessel was monitored for >3 min. Approximately 20 venules from 3mice were monitored for each group of experiments. Data analysis wasperformed using Slidebook 5.5 (Intelligent Imaging Innovations). Toquantify fluorescence signals, fluorescence intensities of albuminnanoparticles internalized into neutrophils were integrated using thesoftware, and the distribution of nanoparticle uptake in neutrophils wasanalyzed.

Statistical Analysis

Data are expressed as mean±SD or SEM. Data were analyzed using one-wayANOVA (multiple groups) or Student t-test (two groups) of Origin 8.5,with p values <0.05 were considered significant.

Results

Uptake of Albumin Nanoparticles by Adherent Neutrophils in Venules.

Intravital microscopy of mouse cremaster muscle venules demonstratedthat Cy5-loaded albumin nanoparticles are internalized by activatedneutrophils following administration of TNF-α (0.5 μg/mouse) and duringsurgical stress-induced vascular inflammation. Neutrophils werevisualized by intravenous infusion of Alexa Fluor 488 anti-mouse Gr-1antibody. In the TNF-α challenge group, the nanoparticles wereintravenously infused 3 hr post-intrascrotal injection of TNF-α (0.5μg/mouse). Monocytes were visualized by infusion of Alexa Fluor 488anti-mouse F4/80 antibody 3 hr after infusion of TNF-α. FIG. 1 shows thepercentage of neutrophils and monocytes internalizing albuminnanoparticles. In all experiments, 100 μg fluorescent dye-labeledalbumin nanoparticles/mouse was infused intravenously. All datarepresent mean ±SEM (n=13-20 vessels in 3 mice per group).

Characteristics of Internalization Properties of Different Types ofAlbumin Nanoparticles.

Three different formulations of fluorescently-labeled albuminnanoparticles were studied. Albumin nanoparticles made from denaturedalbumin were either loaded with Cy5 to form dye-loaded Alb nanoparticlesor the albumin nanoparticle surface was chemically conjugated with AlexFluor 647 or Cy5 to form dye-conjugated Alb nanoparticles, as describedabove. The third nanoparticle formulation was prepared by conjugatingnative (undenatured) albumin to polystyrene nanoparticles having adiameter of 100 nm. Intravital microscopy was performed in mice asabove. Dye-loaded and dye-conjugated albumin nanoparticles wereinternalized by Gr-1-positive neutrophils. Native albumin-conjugatedpolystyrene nanoparticles were bound to the neutrophil surface, and notinternalized. Cy5-conjugated native albumin was not internalized byGr-1-positive neutrophils. With reference to FIG. 2, quantitativeanalysis of percentage of Gr-1-positive neutrophils internalizing thethree types of nanoparticles and Cy5-labeled native albumin shows thatboth dye-loaded Alb nanoparticles and dye-conjugated Alb nanoparticleswere internalized by Gr-1-positive neutrophils, whereas native albuminconjugated polystyrene nanoparticles and Cy5-labeled native albumin werenot internalized by Gr-1-positive neutrophils . Results are shown asmean±SEM (n=13-20 vessels in 3 mice per group). ND=not detected.

Contribution of FcγRIII Mechanism in Mediating Albumin NanoparticleInternalization.

Intravital microscopy of cremaster muscle inflamed venules was performedin wild-type (WT) and FcγRIII^(−/−), Mac-1⁻⁻⁻, and LFA-1^(−/−) mice.Cy5-loaded albumin nanoparticles were intravenously infused 3 hr afterintrascrotal injection of TNF-α (0.5 μg/mouse). Histograms of Cy5-loadedalbumin nanoparticles internalized by neutrophils (more than 500neutrophils in 3 mice per group) in WT and FcγRIII^(−/−) mice showedgreater internalization of Cy5-loaded albumin nanoparticles by WTneutrophils than by FcγRIII^(−/−) neutrophils, indicating a role forFcγRIII in internalization of the albumin nanopoarticles. Similarexperiments performed using Mac-1^(−/−) and LFA-1^(−/−) mice indicatedthat Mac-1 and LFA-1 do not play a role in internalization of thealbumin nanoparticles. FIG. 3 shows the percentage of albuminnanoparticle uptake by polymorphonuclear neutrophils (PMNs) in WT versusknockout mice in each group. P<0.0001 vs. WT mice after ANOVA andDunnett's test. NS, not significant.

Therapeutic Activity of Albumin Nanoparticles in Vascular Inflammationand Lung Injury Models.

Intravital microscopy performed on mice intravenously infused with AlexaFluor 488-conjugated antibodies before and at 1 hr post-intravenousinfusion of piceatannol-loaded albumin nanoparticles (50 μM piceatannol)showed rolling and adhesion of neutrophils. Quantification of neutrophiladhesion and rolling is shown in FIG. 4A and FIG. 4B. FIG. 4A is graphshowing the number of adherent and rolling neutrophils per field inTNF-α-activated cremaster muscle vessels at baseline and at 30 and 60min after intravenous infusion of piceatannol-loaded albuminnanoparticles. FIG. 4B is graph showing the number of adherent androlling neutrophils per field in TNF-α-activated cremaster musclevessels at baseline and at 30 and 60 min after intravenous infusion ofalbumin nanoparticles without piceatannol.

FIG. 5 shows the results of an assay of mouse neutrophils pre-treatedwith 800 μg/ml albumin nanoparticles (NP) or piceatannol-loaded albuminnanoparticles (Pic-NP, 200 μM as piceatannol), and stimulated withN-formyl-methionyl-leucyl-phenylalanine (fMLF). Flow chamber assay atfixed shear representing venous shear (1 dyne/cm²) was performed asdescribed in The number of adherent neutrophils (either spread or round)was quantified during the 10-min recording period. Data representmean±SD (n=3).

Mouse neutrophils were plated on fibrinogen-coated surfaces andincubated with RPMI culture media, 800 μg/ml albumin nanoparticles (NP)or piceatannol-loaded albumin nanoparticles (Pic-NP, 200 μM) in thepresence or absence of 50 ng/ml TNF-α for 30 min. Cell lysates wereimmunoblotted with anti-phospho Syk-Tyr525/526 antibody (FIG. 6). Theresults show that piceatannol inhibited phosphorylation of Syk in TNF-αstimulated neutrophils. Data represent mean±SD (n=3). ** P<0.01 and ***P<0.001 vs. unstimulated cells after ANOVA and Dunnett's test.

Unstimulated or fMLF-stimulated mouse neutrophils were treated with 800μg/ml albumin nanoparticles (NP) or piceatannol-loaded albuminnanoparticles (Pic-NP, 200 μM as piceatannol) for 1 hr. MTT assay wasperformed as described in Methods. Cell viability is presented asmean±SD (n=3). ** P<0.01 vs. unstimulated cells after ANOVA andDunnett's test. Cell viability was not different among any experimentalgroup.

Effects of piceatannol-loaded albumin nanoparticles on LPS-induced lunginflammation were evaluated. The line shown above FIGS. 7A-C describesthe protocol. Lung myeloproxidase (MPO) activity (FIG. 7A) and number ofneutrophils sequestered in lungs (FIG. 7B) before and after intravenousinfusion of piceatannol-loaded albumin nanoparticles is shown. Datarepresent mean±SD (n=3). * P<0.001 vs control after ANOVA. FIG. 7C showsthe concentration of leukocytes in bronchoalveolar lavage (BAL) afterintravenous infusion of albumin nanoparticles (Alb-Nano) orpiceatannol-loaded albumin nanoparticles (Pic-Alb Nano) at a piceatannoldoses of at 4.3 mg/kg body weight. * P<0.01 and ** P<0.05. FIG. 8compares the efficacy of intravenous infusion of piceatannol(Pic)-loaded albumin nanoparticles compared to free piceatannol, at adose of 4.3 mg/kg body weight, in reducing neutrophil infiltration inLPS-induced acute lung inflammation, as assessed by MPO activity. *P<0.05 vs. free piceatannol after Student t-test.

While the disclosure is susceptible to various modifications andalternative forms, specific exemplary embodiments of the presentinvention have been shown by way of example in the drawings and havebeen described in detail. It should be understood, however, that thereis no intent to limit the disclosure to the particular embodimentsdisclosed, but on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the scope ofthe disclosure as defined by the appended claims.

1. A composition comprising nanoparticles, the nanoparticles comprisingdenatured, cross-linked albumin and an agent selected from a therapeuticagent and a detectable moiety, the nanoparticles being capable ofselectively binding to and being internalized by activated neutrophils.